Copyright ß Physiologia Plantarum 2006, ISSN 0031-9317
Physiologia Plantarum 127: 111–118. 2006
ASR1, a stress-induced tomato protein, protects yeast from
osmotic stress
Mariana Bermúdez Morettia,†, Laura Maskinb,†, Gustavo Gudesblatb, Susana Correa Garcı́aa and
Norberto D. Iusemb,*
a
Departamento de Quı́mica Biológica, Facultad de Ciencias Exactas y Naturales, Ciudad Universitaria (1428) Buenos Aires, Argentina
Laboratorio de Fisiologı́a y Biologı́a Molecular, Departamento de Fisiologı́a, Biologı́a Molecular y Celular and IFIBYNE-CONICET, Facultad de Ciencias
Exactas y Naturales, Ciudad Universitaria (1428) Buenos Aires, Argentina
b
Correspondence
*Corresponding author,
e-mail: [email protected]
Received 7 October 2005; revised 15
November 2005
doi: 10.1111/j.1399-3054.2006.00664.x
Asr1, a tomato gene induced by abiotic stress, belongs to a family, composed
by at least three members, involved in adaptation to dry climates. To understand the mechanism by which proteins of this family seem to protect cells
from water loss in plants, we expressed Asr1 in the heterologous expression
system Saccharomyces cerevisiae under the control of a galactose-inducible
promoter. In a mutant yeast strain deficient in one component of the stressresponsive high-osmolarity glycerol (HOG) pathway, namely the MAP kinase
Hog1, the synthesis of ASR1 protein restores growth under osmotic stress
conditions such as 0.5 M NaCl and 1.2 M sorbitol. In contrast, the rescuing
of this phenotype was less evident using a wild-type strain or the upstream
MAP kinase kinase (Pbs2)-deficient strain. In both knock-out strains impaired
in glycerol synthesis because of a dysfunctional HOG pathway, but not in
wild-type, ASR1 led to the accumulation of endogenous glycerol in an
osmotic stress-independent and unrestrained manner. These data suggest
that ASR1 complements yeast HOG-deficient phenotypes by inducing downstream components of the HOG pathway. The results are discussed in terms
of the function of ASR proteins in planta at the molecular and cellular level.
Introduction
Plants constantly have to cope with abiotic stress. Water
deficit, salty soils and cold are the common stress conditions affecting plant development. Among them, high
salinity is one of the most serious limiting factors in
plant growth and productivity (Boyer 1982). In response
to environmental stress, most plants produce an array of
proteins that lead to adaptation of cell metabolism to
these conditions (Ingram and Bartels 1996; Thomashow
1998, 1999).
Our interest is focused in one of the many stressinduced proteins occurring in plants, named ASR1,
encoded by a gene first reported in tomato (Iusem
et al. 1993; Rossi and Iusem 1994). Rossi et al. (1996)
demonstrated that Asr1 actually belongs to a gene
family composed by at least three genes (Asr1, Asr2
and Asr3). Since then, several homologs were isolated
from many different species (reviewed by Maskin et al.
2001).
It is known that tomato Asr1 is expressed under salt
and osmotic stress (Amitai-Zeigerson et al. 1995) and
differentially induced by water deficit (Maskin et al.
2001). ASR1 protein is highly hydrophilic as a consequence of its content of charged residues, particularly
Abbreviations – HOG, high-osmolarity glycerol; LEA, late embryogenesis-abudant; SC, synthetic complete.
†
These authors contributed equally to this work.
Physiol. Plant. 127, 2006
111
His, Lys and Glu (Amitai-Zeigerson et al. 1995; Iusem
et al. 1993) and binds to a specific DNA sequence
(Kalifa et al. 2004), in agreement with evidence in
favour of its localization in the nucleus (Iusem et al.
1993; Wang et al. 2005). It is worth mentioning that
the high hydrophilicity, along with a robust water stress
induction in vegetative tissues, is feature shared by ASR
(Maskin et al. 2001) and most proteins of the late embryogenesis-abundant (LEA) superfamily (Skriver and
Mundy 1990), expressed at high levels during late
embryo maturation (Chung et al. 2005). Although the
biochemical function of ASR1 remains unclear, its
nuclear localization and DNA-binding activity are in
good agreement with the proposed role of ASR as a
regulatory transcription factor involved in the activation
of sugar transport (Cakir et al. 2003).
The baker’s yeast Saccharomyces cerevisiae is a welldeveloped and widely used model organism (Ton and
Rao 2004; Wheeler and Grant 2004) as it offers a compact and fully sequenced genome, simple culturing conditions and a conservation of basic signal transduction
pathways with higher eukaryotes. We decided to take
advantage of this model organism to investigate if there
is any osmoregulatory response mediated by ASR1, in
an attempt to outline the in vivo function of tomato
ASR1. Therefore, the intrinsic physiological response
of S. cerevisiae to an osmotic challenge was to be
taken into account.
Adaptation to osmotic stress in yeast is controlled by
the high-osmolarity glycerol (HOG) MAP kinase pathway (Brewster et al. 1993). This pathway consists of two
transmembrane osmosensors, Sho1p and Sln1p, a
branched MAPK cascade and common downstream elements Pbs2p MAPKK and Hog1p MAPK. A high osmolarity estimulus causes rapid nuclear accumulation of
Hog1p, the sole terminal MAPK (Gustin et al. 1998;
Mager and Siderius 2002). Activated MAPKK Pbs2 phosphorylates the MAPK Hog1p by dual phosphorylation.
Once activated and after translocation to the nucleus
(Sharma and Mondal 2005), Hog1p modulates the
expression of several osmo-inducible genes such as
GPD1, encoding glycerol 3-phosphate dehydrogenase
(Albertyn et al. 1994; Ferrigno et al. 1998; Lee et al.
2002; Reiser et al. 1999). This enzyme is involved in the
synthesis of glycerol, which accounts for the major
compatible solute of Saccharomyces. By increasing
endogenous glycerol, cells regain turgor. Importantly
to this work, the HOG pathway seems to be structurally
and functionally conserved in plants (Covic et al. 1999).
To test the possible osmoprotective function of ASR1,
we forced expression of this protein in the heterologous
system S. cerevisiae under osmotic stress conditions.
We demonstrated that ASR1 is able to rapidly improve
112
yeast cell growth under saline stress conditions.
Moreover, we found that ASR1 is able to complement
osmotolerance deficiencies in hog1D and pbs2D
mutants concomitantly with an increased accumulation
of the osmolyte glycerol.
Materials and methods
Yeast strains and growth conditions
S. cerevisiae strains used in this study are the wild-type
YPH102 (MATa ura3-52 lys2-801amber ade2-101ochre
his3-D200 leu2-D1) and the isogenic mutants JBY13
[YPH499 (MATa ura3-52 lys2-801amber ade2-101ochre
trp1-D63 his3-D200 leu2-D1) þ hog1D::TRP1] and
MAY1 (YPH102 þ pbs2D::LEU2). Yeast was grown at
30 C in YPD medium (1% yeast extract, 2% peptone
and 2% glucose) or in synthetic complete (SC) medium
(0.17% yeast nitrogen base without amino acids and
ammonium sulphate supplemented with Ura-Dropout
and 10 mM ammonium sulphate). Carbon sources for
synthetic medium were 2% raffinose or 1% galactose.
Stress assays
Yeast was cultivated in SC medium containing 2% raffinose for 24 h. SC media containing 2% raffinose or 1%
galactose were supplied with 1/5 volume of the prepared preculture, and cells were incubated for different
periods of time as indicated in the figures. Cells were
then subjected to osmolarity stress at various NaCl concentrations, and growth was determined by measuring
absorbance at 600 nm.
To test osmosensitivity in solid medium, we grew
cells in 2% raffinose SC for 24 h, then transferred to
1% galactose SC and incubated for 3 h. Serially diluted
(1:10) cell suspensions (3 ml) were spotted on YPD
plates supplemented with various concentrations of
NaCl or sorbitol. After three days at 30 C, plates were
photographed.
Construction of the recombinant vector
All DNA manipulations were performed by standard
procedures (Sambrook et al. 1989). The tomato Asr1
full-length cDNA was PCR amplified using primers containing Xba1 and Kpn1 restriction sites (upper primer:
5’-GCTCTAGACTAAAATAACAATTTAGAAGAGAT-3’;
lower primer: 5’GGGGTACCATGGAGGAGGAGAAACACCACCAC-3’). The cDNA was then cloned into the
Xba1/Kpn1 site of the high-copy-number shuttle vector
pYES2 (Invitrogen, CH Groningen, the Netherlands) that
contains the GAL1 promoter. Wild-type and mutant
Physiol. Plant. 127, 2006
strains were transformed with the resulting recombinant
vector using the method described by Chen et al.
(1992). As a control, the same strains were transformed
with the empty vector.
Protein extraction and Western blot assay
Yeast total protein was extracted with a buffer containing 0.06 M Tris HCl pH 6.8, 5% (v/v) b-mercaptoethanol, 10% glycerol, 2% sodium dodecyl sulphate (SDS).
Western blots were performed according to Sambrook
et al. (1989), loading 20 mg of total protein. After SDSPAGE, proteins were electroblotted onto Hybond-C
Extra nitrocellulose membrane (Amersham Pharmacia
Biotech, Piscataway, NJ, USA). Membranes were
stained with Ponceau red to ensure equal loading and
then incubated with ASR1-specific antiserum (1/4000).
Bound antibodies were detected using an anti-rabbit
immunoglobulin G-peroxidase conjugate (Biorad,
Hercules, CA, USA) diluted 1/4000. For visualization
of enzyme activity, the ECL chemiluminescence detection system was used (Amersham Pharmacia Biotech).
Glycerol measurements
Cells from an overnight culture in 2% raffinose SC medium were transferred to fresh SC medium containing 2%
raffinose or 1% galactose and incubated for 1 h. Cells
were then transferred again to similar fresh media in the
presence or absence of 0.25 M NaCl. After 3 h of incubation, two 10-ml samples were taken out for glycerol
and total protein measurements. Cells were pelleted,
washed with cold water and centrifuged again at 4 C.
For glycerol determinations, pellets were resuspended
in 1 ml 50 mM Tris-HCl pH 7.5, boiled for 15 min and
centrifuged to remove cellular debris. The supernatant
was used for glycerol quantification according to the
manufacturers’ instructions (glycerol measurement kit,
Boehringer Mannheim, Mannheim, Germany).
Protein content was determined by the Bradford
method (Bradford 1976).
Results
Effect of ASR1 expression on yeast growth
To test the possible role of ASR1 in protecting cells from
water loss, we chose S. cerevisiae as a convenient heterologous expression host system. The wild-type strain
was transformed with the plasmid pYES2 containing the
Asr1 clone driven by the galactose-inducible promoter
GAL1. Cells transformed with the empty vector were
used as a control. Cells derived from inducible conditions were transferred to fresh 1% galactose media containing or not 0.5 M NaCl. ASR1 seemed to confer an
advantage in cell survival at early times of exposure to
the high saline environment (Fig. 1). Under normal conditions, a detrimental effect of ASR1 in cell growth was
observed (data not shown), as also reported for other
heterologous proteins overexpressed in yeast (Choi et al.
1994; Kiel et al. 1999). The slight protective effect of
ASR1 on wild-type cell growth under saline stress conditions was detected only at early times probably due to
the establishment of yeast intrinsic adaptation mechanisms as time passes by. It should be noted that the
observed behaviour may be the outcome of both a
positive osmoprotective effect and the simultaneous
and detrimental effect brought about by ASR1.
The presence of ASR1 in the culture was confirmed
by Western blot (Fig. 1, inset).
120
*
.
60
40
20
pYES2-Asr1
80
pYES2
% Survival
100
-ASR1
0
0
2
4
Time (h)
Physiol. Plant. 127, 2006
Fig. 1. Effect of ASR1 on yeast survival
under salt stress. YPH102 cells transformed
with pYES2 (*) or pYES2-Asr1 ( ) were
grown on 1% galactose medium for 17 h
and then transferred (t 5 0) to fresh 1%
galactose synthetic complete medium supplemented or not with 0.5 M NaCl, and
absorbance at 600 nm (A600nm) was
determined at the indicated times. Survival
percentage was expressed as 100 · A600nm
of the culture with NaCl/A600nm of the
culture without NaCl. Values presented are
the mean of three independent experiments.
Error bars denote mean standard error.
*, P value < 0.05 (according to student’s
t-test). Inset: total protein extracts from
YPH102 cells at 0 min were analysed by
Western blot using a specific ASR1
antiserum.
6
8
113
As high expression levels of ASR1 in wild-type yeast
cells produced some deleterious effect on cellular
growth, we decided to diminish intracellular ASR1
amounts by shortening the time of induction by galactose. Total protein was extracted from cultures induced
with galactose for different periods of time and analysed
by immunoblot (Fig. 2). Although the maximum expression levels were reached after 8 h of induction, 3 h
were enough to obtain a significant ASR1 expression.
Consequently, cells preinduced for 3 h were plated
on complete media with different NaCl concentrations
and assayed for growth. An overall detrimental effect
caused by high osmolarity could be observed (Fig. 3,
first row of each panel). The expression of ASR1 resulted
in a slight, but consistently observed, improved salt
resistance of wild-type cells (Fig. 3). It must be noted
that this result was obtained after 3 days of growth in
non-inductive conditions. The turnover of the heterologous protein was slow enough to allow its immunodetection even 48 h after the induction period (Fig. 4).
NaCl concentrations higher than those indicated
allowed no growth at all even in Asr-bearing cells (not
shown).
Functional analysis of ASR1 in yeast
To get further insight into the osmoprotective function of
ASR1 in yeast cells, the effect of this protein was analysed in cells deficient in the HOG pathway, which bear
a phenotype markedly sensitive to osmotic stress due to
their inability to synthesize normal amounts of glycerol
(Brewster et al. 1993). ASR1 turned out to provide protection to hog1D cells, as it allowed them to grow at
0.5 M NaCl, a concentration otherwise lethal for this
strain (Fig. 5). However, we failed to detect ASR1 overcoming the growth impairment of salt-stressed pbs2D
cells (Fig. 5), even when different stress conditions (not
shown) were used (0.6 and 0.75 M NaCl; 0.5, 0.6 and
0.75 M KCl; 0.9 M sorbitol). A possible explanation is
0
3
6
8
18
25
(h)
-ASR1
Fig. 2. Analysis of ASR1 expression levels in yeast cells after different
times of galactose induction. YPH102 cells transformed with pYES2Asr1 were grown on 2% raffinose synthetic complete (SC) medium and
transferred (t 5 0) to 1% galactose SC medium. At the indicated times,
total protein extracts were prepared and analysed by Western blot.
114
Wild type
pYES2
YPD
pYES2-ASR1
pYES2
YPD 0.5 M NaCl
pYES2-ASR1
pYES2
YPD 0.9 M NaCl
pYES2-ASR1
Fig. 3. Effect of ASR1 on yeast growth under different saline concentrations. YPH102 cells transformed with pYES2 or pYES2-Asr1 were
grown on 2% raffinose synthetic complete (SC) medium and then
incubated for 3 h with 1% galactose. Serially diluted (1:10, starting
A600nm 5 0.1) cell suspensions (3 ml) were spotted on YPD plates without or with 0.5 M NaCl and 0.9 M NaCl. After 3 days at 30 C, plates
were photographed.
that the higher amounts of endogenous ASR1 found in
the latter mutant (Fig. 4) may be the cause of its poor
performance. It is noteworthy that although the number
of colonies for this mutant did not change, they became
smaller in size in the presence of an osmolyte, indicating a lower number of cell divisions and thus the occurrence of stress.
We also incubated yeast mutant cells in a medium
containing sorbitol, a non-charged osmolyte. The resulting effect was in the same direction but slighter than that
caused by NaCl (Fig. 5, bottom panels), suggesting that
the change in cellular ion balance is an important, but
not exclusive, component of the overall stress against
which Asr protects.
These results prompted us to determine intracellular
glycerol content in cells challenged to stress. The stress
conditions used were mild (0.25 M NaCl) to allow normal growth of the deficient yeast strains. In wild-type
cells (Figs. 6A and B), accumulation of glycerol
depended on the presence of NaCl, irrespective of the
expression of ASR1. Interestingly, the expression of
ASR1 in hog1D cells produced a significant accumulation of glycerol, and this effect was independent on the
imposition of any salt stress (Fig. 6C). Similar results
were observed in pbs2D cells, although the glycerol
content was lower than that measured in hog1D cells.
The results indicate that ASR1 produces an important
increase in glycerol accumulation only when the HOG
pathway is dysfunctional. In contrast, when this pathway is normally operating, ASR1 per se does not modify
Physiol. Plant. 127, 2006
hogΔ
Wild type
0
6
48
0
6
pbs2Δ
48
0
6
48
(h)
-ASR1
Fig. 4. Life time of ASR1 protein in yeast cells. Wild-type (YPH102), hog1D (BJY13) and pbs2D (MAY1) cells carrying pYES-Asr1 were grown on 2%
raffinose synthetic complete (SC) medium and then transferred to 1% galactose SC medium. After 3 h of incubation, cells were transferred to YPD
medium (t 5 0) and incubated at 30 C. Samples were withdrawn at the indicated times, and total protein extracts were prepared and analysed by
Western blot.
the glycerol phenotype, suggesting a tight regulation of
glycerol content by the HOG pathway.
Discussion
Asr1 is one of the numerous plant gene products that
become overexpressed under water and salt stress conditions (Ramanjulu and Bartels 2002). On the other
hand, yeast cells are considered an attractive eukaryotic
model expression system used to address the function of
plant genes (Cadinanos et al. 2003; Hashimoto et al.
2004; Yamaguchi et al. 2003; Zhuang et al. 2005).
Inasmuch as plant responses to environmental salt and
water deficit stress have much in common, because
salinity reduces the ability of plants to take up water
(Munns 2002), we expressed the water deficit stressinduced tomato Asr1 gene in the S. cerevisiae host
under osmotic stress to gain insights into the in planta
function of ASR1 protein.
The inability of pbs2D and hog1D to grow in the
presence of high salt concentrations conforms the previously observed dramatic reduction of osmoresistance
of such yeast mutant cells lacking a functional HOG
pathway (Brewster et al. 1993). Interestingly, the
hog1Δ
pbs2Δ
pYES2
YPD
pYES2-ASR1
pYES2
YPD 0.5 M NaCl
pYES2-ASR1
pYES2
YPD 1.2 M Sorbitol
pYES2-ASR1
Physiol. Plant. 127, 2006
osmoprotective effect of ASR1 turned out to be more
evident in such yeast strains. In fact, our results demonstrate that ASR1, in spite of being a source of mild stress
itself, is able to complement the growth impairment
phenotype inherent to the hog1D mutant. Although
both strains are defective in the same pathway, the
absence of the terminal kinase Hog1p leads to a stronger osmosensitivity than that observed in the absence of
the kinase kinase Pbs2p. Although many stress conditions were tested, we found no overt restoring of pbs2D
growth by ASR1. However, such a scenario could be
foreseen, if more severe but not lethal stress conditions
were imposed.
Yeast cells respond to a shift to higher osmolarity by
increasing the cellular content of the osmolyte glycerol
(Shen et al. 1999). In this context, the mere production
of heterologous proteins might as well trigger broad
stress responses, including synthesis of glycerol and
would thus lead to misinterpretation of the results associated with osmoprotection. Nevertheless, this was not
the case as ASR1 alone produced no increase in the
accumulation of glycerol in wild-type cells. The puzzling fact that hog1D cells are able to produce substantial amounts of glycerol in basal conditions is in good
Fig. 5. Effect of ASR1 on
growth of HOG pathwaydefective mutants under osmotic
stress. Wild-type (YPH102),
hog1D (JBY13) and pbs2D
(MAY1) cells transformed with
pYES2 or pYES2-Asr1 were
grown on 2% raffinose synthetic complete (SC) medium,
then transferred to 1% galactose
SC medium. After 3 h of
incubation, serially diluted (1:10,
starting A600nm 5 1.0) cell
suspensions (3 ml) were spotted
on YPD plates and on YPD
plates with 0.5 M NaCl or 1.2 M
sorbitol. After 3 days at 30 C,
plates were photographed.
115
µg glycerol/mg protein
600
A
500
400
300
200
100
0
600
B
500
400
300
200
100
0
600
500
C
400
300
200
100
0
600
D
500
400
300
200
100
0
Raffinose
Raffinose +
NaCL
Galactose Galactose +
NaCL
Fig. 6. Intracellular glycerol levels in yeast cells. Cells from an overnight culture in 2% raffinose synthetic complete (SC) medium were
transferred to fresh SC medium containing 2% raffinose or 1%
galactose and incubated for 1 h. Then, cells were transferred again
to similar fresh media containing or not 0.25 M NaCl. After 3 days
of incubation, two 10-ml samples were taken for glycerol and
protein quantification. Standard deviations were lower than 15%.
(A) YPH102 wild-type cells transformed with empty plasmid
(pYES2). (B) YPH102 wild-type cells transformed with the recombinant plasmid (pYES2-Asr1). (C) JBY13 cells (hog1D) transformed
with the recombinant plasmid (pYES2-Asr1). Glycerol value for
control cells with empty plasmid induced with galactose was
110 mg glycerol/mg protein. (D) MAY1 cells (pbs2D) transformed
with the recombinant plasmid (pYES2-Asr1). Glycerol value for
control cells with empty plasmid induced with galactose was
88 mg glycerol/mg protein.
116
agreement with the findings by Albertyn et al. (1994),
who postulated a hog1-independent mechanism mediating osmostress-induced glycerol accumulation. If this
putative alternative pathway indeed exists, it seems to
be more active in the mutant cells than in the wild-type
genotype, probably because hog1p normally inhibits
shared upstream components of the HOG pathway, as
part of a feedback control on the signal cascade, as
envisaged by O’Rourke and Herskowitz (1998).
Only in mutant strains bearing a dysfunctional HOG
pathway, ASR1 led to the accumulation of glycerol in an
osmotic stress-independent manner. The mechanisms
by which ASR1 restores adequate intracellular glycerol
levels in such an unrestrained fashion are still obscure to
understand. The complementation experiments in yeast
suggest a genetic mode of action for ASR1 by inducing
downstream components of the HOG pathway, which
is known to exist in plants as well (Covic et al. 1999).
However, based on its abundant expression in several
plant tissues and its high degree of hydrophylicity
(Iusem et al. 1993; Maskin et al. 2001), an alternative,
but not mutually exclusive, structural function such as
the promotion of direct protection from water loss cannot be ruled out, as is the case for a mitochondrial LEA
protein able to protect enzymes from drying (Grelet
et al. 2005). Independently of what the mechanism
might be, the involvement of ASR proteins in physiological adaptation of wild tomato to dry climates is
strongly supported by our studies through evolutionary
approaches concluding that Asr2 has been the target of
positive natural selection (Frankel et al. 2003).
Very recently, ASR from lily pollen has been reported to
confer drought and salt resistance in Arabidopsis (Yang
et al. 2005), although no precise physiological mechanism could be concluded. Current research using transgenic plants is in progress in our laboratory with the
intention to fully understand the molecular and cellular
mechanism of action of these widespread proteins underlying adaptation of plants to environmental stress.
Acknowledgements – This research was supported by
University of Buenos Aires (UBA) and Consejo Nacional
de Investigaciones Cientı́ficas y Tecnológicas (CONICET),
Argentina. The authors thank Lic. Nicolás Frankel and
Dr Alejandro Colman Lerner for helpful discussions on the
manuscript.
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